The focus of this Combined Clinical and Technology Research Site (CTRS) application is to apply novel histomorphometric and transcriptome analytics to define the interplay between lymphocytes (B cells and T cells) and bone homeostasis in rheumatoid arthritis (RA) within target tissue and well-characterized patient populations. We plan to develop and optimize strategies to categorize subsets of disease, define biomarkers of treatment response, and identify novel treatment targets. Research to date in RA has focused on defining abnormalities in individual immune cell populations or signaling pathways involved in osteoclast (OC) differentiation and activation or osteoblast (OB) function, but very few groups have provided an integrated assessment of the multiple relevant cell populations in the disease and the interactions between the lymphocyte and bone compartments in target tissue. Although prior approaches have yielded important clues regarding immune pathogenesis, major limitations in the field include the lack of biomarkers to target appropriate treatment strategies and the failure of currently available treatments to achieve low disease activity and/or limit progressive joint damage in the majority of patients. Our group has made multiple observations linking B cells to joint damage including the efficacy of B cell depletion as a treatment and the presence of B cell aggregates in rarely studied target tissue including the synovium, subchondral bone, and bone marrow (BM) where OBs and OCs develop, associated with T cell and OC activation and more recently OB dysfunction. Additional seminal observations by our group have defined monocyte dysfunction in RA and characterized key down-stream signaling pathways in OC differentiation and activation. However, the role and precise mechanisms of aberrant B-T cell interactions and other pathways (including monocyte and cytokine activation) in mediating joint destruction in the target tissue are relatively unexplored and a ripe area for identification of new treatment targets. This assessment to date has been hampered by lack of access to relevant target tissue and the need for novel technologic approaches to better define cellular and cytokine networks. We can surmount these limitations given a unique collaboration between Rheumatology and Orthopedics with access to unprecedented surgical and biopsy samples from untreated RA patients and powerful technologies within the Center for Musculoskeletal Research (CMSR) at the University of Rochester (UR) already well-validated for the study of musculoskeletal diseases. In this proposal we will (1) Integrate clinical, laboratory, and ultra-sound (US) data on treated and untreated RA patients and establish optimal methods for acquisition of synovial tissue by US-guided needle biopsy, (2) Characterize the localization of relevant immune and bone cells in the joint using novel histomorphometric tools for automated quantitation, and (3) Define cell function by RNA sequencing of discrete sorted and captured cell populations in synovium. The powerful dual approach of characterizing the location of key cell populations at the bone-pannus junction and defining cell function within this 3D milieu using novel histomorphometric tools and RNA sequencing of discrete cell populations will better reflect underlying disease pathogenesis, allowing more precise patient stratification and identification of novel targets.